Protease inhibitors derived from guamerin

ABSTRACT

The present invention relates to peptides inhibiting elastase and subtilisin activity, which are derived from Guamerin, an elastase-inhibiting protein isolated from a Korean leech, Guameri(Hirudo nipponia). Since the peptides of the invention permit their convenient synthesis and use, it can be applied for the development of elastase- and subtilisin-inhibiting agents. Also, since the dimeric peptides of the invention have strong elastase- and subtilisin-inhibiting activities, they can be more practically applied for the treatment of diseases associated with elastase and subtilisin. Moreover, all of the peptides of the invention can be safely used for human body as a potential drug, since they have relatively lower molecular weights.

This is a 35 U.S.C. § 371 application of PCT/KR97/00036, filed Mar. 11,1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to peptides derived from Guamerin whichinhibit protease activity, more specifically, to peptides inhibitingelastase and subtilisin activity, which are derived from Guamerin, anelastase-inhibiting protein isolated from a Korean leech, Guameri(Hirudonipponia).

2. Description of the Prior Art

Elastase is a serine protease capable of degrading mainly elastin andalso connective tissue proteins such as collagen, cartilage, andfibronectin(see: Reilly, C. et al., Biochem. Biophys. Acta.,621:147-167(1980); Mainardi, C. L. et al., J. Biol. Chem.,255:5436-5441(1980)).

Human leukocyte elastase is stored principally in neutrophils and thestored elastase is released, when neutrophils encounter foreignpathogens or antigens in blood, to degrade them so that body isprotected from the harmful factors(see: Weisemann, G. et al., New Engl.J. Med., 303:27-34(1980)). However, uncontrolled secretion of elastasewhich frequently results from aging of the cells or genetic disorder maycause non-specific proteolysis and trigger destructive processesassociated with various chronic diseases such as rheumatoid arthritis,emphysema, and psoriasis(see: Glinski, W. et al., J. Invest. Dermatol.,75:481-487(1980); Snider, G. L., Med. Clin. North. Am.,65:647-666(1981)).

In medical field, for the treatment of said diseases, strenuous effortshave been made in developing an agent which can effectively suppress theactivity of elastase which is released abnormally in excess from thetissues of joint cartilage, lung, and skin. As a consequence,elastase-inhibiting proteins have been isolated from a variety ofbiological sources such as birds including turkeys or ducks, Europeanleeches, and human skin(see: Schalwijk, J. et al., Br. J. Dermatol.,1512:181-186(1986).; Wlodow, O. et al., J. Biol. Chem.,165:14791-14796(1990); Hochstrasser, K. et al., Hopps-Seyler's Z.Physiol. Chem., 362:1369-1375(1981); Seemiller, U. et al.,Hopps-Seyler's z. Physiol. Chem., 361:1841-1846(1980)), which were foundto be effective for the treatment of said diseases, especially when amedicine containing the protein as an active ingredient was administereddirectly to the affected parts.

However, the elastase-inhibiting proteins of prior art, except the oneisolated from human skin, have had trouble for the use as a medicine,since their specificity for elastase is so low that the activities ofother enzymes are possibly inhibited. Moreover, since the saidelastase-inhibiting proteins including the one from human skin have anextremely high molecular weight, a serious problem has been frequentlyencountered that the proteins may be easily denatured by heat, whichfinally decreases their activities rapidly.

Under the circumstances, there are strong reasons for exploring anddeveloping alternative proteins which inhibit the elastase activity in aspecific manner. In this connection, the present inventors have isolateda novel elastase-inhibiting protein named ‘Guamerin’, from a Koreanleech, Guameri(Hirudo nipponia) and discovered that Guamerin inhibitsthe elastase activity specifically, has stronger activity than theconventional elastase-inhibiting proteins, and is also stable understrongly acidic or alkaline condition(see: UK Patent Application GB2300190A; H. I. Jung et al., J. Biol. Chem., 270(23):13879-13884(1995)). Accordingly, Guamerin has a distinction over theconventional elastase-inhibiting proteins that: it may not causeuntoward effects, when it is administrated as a medicine; and, itschemical nature is so stable that it is not easily denatured in thecourse of mass production, storage and transport, which naturally easesits practical application.

However, since Guamerin is still large molecule to be practicallyapplied in medicinal use, the inventors have made an effort to developpeptides which contain active sites of Guamerin showingelastase-inhibiting activities permitting its convenient synthesis anduse.

SUMMARY OF THE INVENTION

In accordance with the present invention, the inventors discovered that:peptides consisting of 19 amino acids which contain the 36th-methionineof the active site of Guamerin, show elastase-inhibiting activities;and, the said peptides, unlike an intact Guamerin, also inhibitsubtilisin activity and elastase activity as well.

A primary object of the invention is, therefore, to provide peptidesderived from Guamerin which inhibit elastase and subtilisin activities.

The other object of the invention is to provide dimeric peptides formedby intermolecular disulfide bond between the peptides, which alsoinhibit elastase and subtilisin activities.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects and features of the present inventionwill become apparent from the following descriptions given inconjunction with the accompanying drawings, in which:

FIG. 1 is the amino acid sequences of Guamerin(SEQ ID NO:1) and twopeptides derived therefrom, i.e., pM and pR(SEQ ID NO:2 and SEQ IDNO:3).

FIGS. 2(A) and 2(B) represent HPLC patterns of two synthetic peptides ofthe invention, i.e., pM and pR, after oxidoreductive reaction.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors purified an elastase-inhibiting protein, i.e.,Guamerin, from fully matured leeches collected in Korea, by a series ofisolation steps including acetone extraction, gel filtration,anion-exchange chromatography and reverse-phase HPLC(high performancelicuid chromatography). Guamerin was found to be a protein of amolecular weight of 6,110 Da which is composed of 57 amino acidresidues(see: FIG. 1) Further, it was determined that: Guamerin contains10 cysteine residues which are required for the formation of disulfidebond permitting rigid structure of a protein; and, its active site isoccupied with a methionine residue at the 36th position and anisoleucine residue at the 37th position, respectively(see: UK PatentApplication GB 2300190A; H. I. Jung et al., J. Biol. Chem., 270(23):13879-13884(1995)).

The present inventors, in order to develop short peptides which affordtheir convenient synthesis and use, while possessing the same activitiesof Guamerin, have synthesized two peptides as followings: a peptide(pM)consisting of 19 amino acids in which the 36th-methionine or the activesite of Guamerin, is Positioned at the center; and, a peptide(pR) havingthe same amino acid sequence as the said pM except for argininesubstituted for the 36th-methionine said peptides may be synthesized bychemical means or by employing DNA manipulation techniques conventionalin the art.

Determination of biochemical activities has proved that: the twosynthesized peptides show inhibitory activities against only elastaseand subtilisin among various protease; and, inhibitory activity of theshort peptide is not changed, even though the 36th-methionin issubstituted with other amino acid. Therefore, it was concluded that thesaid peptides can be used as active ingredients for potential elastase-and subtilisin-inhibiting agents.

On the other hand, since disulfide bond formed by cross-linking ofcysteines may affect protein structure which is critical to theiractivities, oxidoreductive reaction was carried out to form disulfidebonds in the synthesized peptides. Then, the oxidoreduced peptides wereisolated, and their protease-inhibiting activities and molecular weightswere determined. As a result, it was found that dimeric peptides formedby intermolecular disulfide bond between the monomers, by theoxidoreductive reaction, have much stronger elastase- andsubtilisin-inhibiting activities than the monomers. Particularly, it wasfound that the dimeric peptides have elastase-inhibiting activitiesalmost the same as that of Guamerin, and very high subtilisin-inhibitingactivities unlike Guamerin.

Accordingly, since the dimeric peptides of the invention have strongelastase- and subtilisin-inhibiting activities and can be synthesized byconvenient means, they may be more practically applied for the treatmentof diseases associated with elastase and subtilisin, when compared toGuamerin. Moreover, they can be safely used for human body as apotential drug, since they have relatively lower molecular weights.

The present invention is further illustrated in the following examples,which should not be taken to limit the scope of the invention.Particularly, in accordance with the purposes of the invention, allGuamerin-derived monomeric and dimeric peptides havingprotease-inhibiting activities should be fallen within the scope of thepresent invention, in addition to monomeric and dimeric pM and pRdescribed in the following examples which have elastase- andsubtilisin-inhibiting activities.

EXAMPLE 1 Purification and characterization of Guamerin

Fully matured leeches collected in Korea were treated with 95% (v/v)ethyl alcohol to remove stomach impurities and blood clot. Then, theywere put in 80% (v/v) acetone and homogenized to prepare acetoneextract. The acetone extract was concentrated and applied on a SephadexG-75 column(Sigma, USA). After washing the column, fractions showing theelastase-inhibiting activity were pooled and applied on a DEAE-Sepharosecolumn(Sigma, USA). After washing and eluting the column, fractionsshowing the elastase-inhibiting activity were pooled, concentrated andapplied on a reverse-phase HPLC column(Delta-pak C18, Millipore, USA).Thus, Guamerin, an elastase-inhibiting protein was finally purified.

As a result of analyses of molecular weight and amino acid sequence, itwas found that: Guamerin is a protein of molecular weight of 6,100 Dawhich is composed of 57 amino acid residues; and, it contains 10cysteine residues which are required for formation of disulfide bondpermitting rigid structure of a protein(see: FIG. 1). Also, it wasdetermined that the active site of Guamerin includes 36th-methionine and37th-isoleucine, and Guamerin retains an inihibiting-activity highlyspecific to elastase. Moreover, it was found that Guamerin showsstability against heat as well as strong acids and alkalies.

EXAMPLE 2 Chemical synthesis of peptides derived from Guamerin anddetermination of their activities

In order to synthesize short peptides having the same activities ofGuamerin, peptides consisting of 19 amino acids in which 36th-methionineresidue of the active site of Guamerin is located in the center, weredesigned. Then, two peptides, i.e., pM and pR, were synthesizedchemically, based on the amino acid sequence of Guamerin(SEQ ID NO:1)disclosed in FIG. 1. In FIG. 1, pM is a peptide having the amino acidsequence(SEQ ID NO:2) from 31th-threonine to 49th-glycine of Guamerin,and pR is a peptide having the same amino acid sequence as pM except for36th-methionine substituted with arginine(SEQ ID NO:3). The two peptideswere designed so that they contain two cysteines near the active site,which, in turn, provide disulfide bond formed by cross-linking to give adesired active product whose chemical structure was stabilized.

The synthesized peptides were applied on a reverse-phase HPLC column,and purified by elusion with a linear gradient of acetonitrilecontaining 0.1% trifluoroacetic acid. The activities of the purifiedpeptides were determined, based on the inhibitory activities againstdegradation of a substrate, i.e., azocasein, by various kinds ofprotease such as trypsin, chymotrypsin, subtilisin and elastase, whilecontrolling the concentrations of a peptide and a protease at a ratio of20:1 (w/v). As a result, it was found that the synthetic peptides showinhibitory activities against only elastase and subtilisin, whosepercentage of maximum inhibition is 7 to 9%(see: Table 2).

EXAMPLE 3 Determination of activities of peptide dimers formed bydisulfide bond

Since two cysteines of the synthetic peptides may affect the activitiesof the peptides, effects of disulfide bonds in the peptides(i.e., pM andpR), was studied after carrying out oxidoreductive reaction of thecysteine residues. In this connection, the oxidoreductive reaction wascarried out by incubating the peptides in 0.1 M sodium acetate buffer(pH7.8) containing 1 M guanidine hydrochloride with 0.2 mM oxidizedglutathione(GSSG) and 1mM reduced glutathione(GSH) for 2 hours at 37° C.

The oxidoreduced peptides were isolated by employing the HPLC columnused in Example 2(see: FIGS. 2(A) and 2(B)). FIGS. 2(A) and 2(B)represent HPLC patterns of the synthetic peptides, i.e., pM and pR,respectively, after the oxidoreductive reaction. As shown in FIGS. 2(A)and 2(B), there were five peptides derived from the synthetic peptide ofpM, and the peptides corresponding to peaks were named pM(1), pM(2),pM(3), pM(4) and pM(5), respectively. Also, there were seven peptidesderived from the synthetic peptide of pR, and the peptides correspondingto peaks were named pR(1), pR(2), pR(3), pR(4), pR(5), pR(6) and pR(7),respectively.

Then, elastase- and subtilisin-inhibiting activities of the isolatedpeptides were assayed, and molecular types of the peptides weredetermined by the aid of matrix-assisted laser desorptionionization(MALDI) mass spectrometry. The results were summarized inTable 1 below.

TABLE 1 Types and protease-inhibiting activities of syntheticpeptides(*) Inhibitory activity Peptide Molecular type ElastaseSubtilisin pM(1) N.D. N.D. N.D. pM(2) dimer +++ +++ pM(3) dimer +++ +++pM(4) dimer +++ +++ pM(5) monomer + + pR(1) N.D. N.D. N.D. pR(2) N.D.N.D. N.D. pR(3) dimer +++ +++ pR(4) dimer +++ +++ pR(5) monomer + +pR(6) dimer +++ +++ pR(7) monomer + + *N.D.: not detected (+++) and (+)indicate strong and weak activities, respectively.

As can be seen in Table 1, it was found that the peptides showing strong(high) elastase- and subtilisin-inhibiting activities are dimers of pMand pR peptides each of which is derived from the synthetic peptides.Therefore, it was clearly demonstrated that oxidoreductive reaction ofmonomeric peptides pM and pR provides dimers to possess much strongerelastase- and subtilisin-inhibiting activities than the monomers.

Further, in order to compare various protease-inhibiting activities ofthe dimers with those of Guamerin and monomeric peptides derivedtherefrom(i.e., pM and pR), their inhibitory activities against trypsin,chymotrypsin, subtilisin and elastase were determined in the same manneras in Example l(see: Table 2). In Table 2, pM-D and pR-D indicate dimersof pM and pR, respectively, and pM-PE indicates a peptide where -SHgroup of cysteine in pM peptide is pyridylethylated.

TABLE 2 Protease-inhibiting activities or Guamerin and the syntheticpeptides of the invention Percentage of maximum inhibition (%)Inhibitory constant Chymo- Sub- Peptide Elastase Subtilisin TrypsinTrypsin Elastase tilisin Guamerin 82  8 6 7 0.81 fM — pM-D 86 97 N.D.N.D.   49 nM 31 nM pM  7  8 N.D. N.D.  — — pM-PE N.D. N.D. N.D. N.D.  —— pR-O 82 91 N.D. N.D.   54 nM 38 nM pR  8  9 N.D. N.D.  — — *N.D. : notdetected

As can be seen in Table 2, it was found that dimeric peptides have muchstronger elastase- and subtilisin-inhibiting activities than monomers,that is, their percentage of maximum inhibition were determined as 82 to97%, respectively. Particularly, it was determined that dimeric peptideshave elastase-inhibiting activities almost the same as that of Guamerin,and they have very high subtilisin-inhibiting activities unlikeGuamerin.

In addition, in order to obtain inhibitory constants of the dimersagainst elastase, inhibitory activities of the dimers againstdegradation of a chromogenic peptide substrate,N-succinyl-L-Ala-Ala-p-nitroanilide by elastase, were determined. As aresult, it was found that inhibitory constants of- the dimers, i.e.,pM-D and pR-D, were 49 nM and 54 nM, respectively(see: Table 2). Also,inhibitory constants of the dimers against subtilisin were determined inthe same manner using N-succinyl-L-Ala-Ala-Pro-Phe-p-nitroanilide as achromogenic peptide substrate. As a result, it was found that inhibitoryconstants of the pM-D and pR-D were 31 nM and 38 mM, respectively(see:Table 2). Though inhibitory constants of the dimers against elastase andsubtilisin show higher values than that of Guamerin againstelastase(0.81 fM), they show significant values compared to theconventional inhibitory agents.

As clearly illustrated and demonstrated as aboves, the present inventionprovides peptides which inhibit elastase and subtilisin activity, whichare derived from Guamerin isolated from a Korean leech, Guameri(Hirudonipconia). Since the peptides of the invention permit their convenientsynthesis and use, it can be applied for the development of elastase-and subtilisin-inhibiting agents. Also, since the dimeric peptides ofthe invention have strong elastase- and subtilisin-inhibitingactivities, they can be more practically applied for the treatment ofdiseases associated with elastase and subtilisin. Moreover, they can besafely used for human body as a potential drug, since they haverelatively lower molecular weights.

3 57 amino acids amino acid single linear protein unknown 1 Val Asp GluAsn Ala Glu Asp Thr His Gly Leu Cys Gly Glu Lys Thr 1 5 10 15 Cys SerPro Ala Gln Val Cys Leu Asn Asn Glu Cys Ala Cys Thr Ala 20 25 30 Ile ArgCys Met Ile Phe Cys Pro Asn Gly Phe Lys Val Asp Glu Asn 35 40 45 Gly CysGlu Tyr Pro Cys Thr Cys Ala 50 55 19 amino acids amino acid singlelinear peptide unknown 2 Thr Ala Ile Arg Cys Met Ile Phe Cys Pro Asn GlyPhe Lys Val Asp 1 5 10 15 Glu Asn Gly 19 amino acids amino acid singlelinear peptide unknown 3 Thr Ala Ile Arg Cys Arg Ile Phe Cys Pro Asn GlyPhe Lys Val Asp 1 5 10 15 Glu Asn Gly

What is claimed is:
 1. An isolated peptide consisting of the amino acidsequence listed as SEQ ID NO: 2, said peptide having inhibitory activityagainst elastase and subtilisin.
 2. An isolated peptide consistingessentially of the amino acid sequence listed as SEQ ID NO:3, saidpeptide having inhibitory activity against elastase and subtilisin. 3.An isolated dimeric peptide which inhibits elastase and subtilisinactivity, wherein said dimeric peptide is formed by an intermoleculardisulfide bond between two peptides wherein each of the two peptidesconsist essentially of SEQ ID NO:
 3. 4. An isolated dimeric peptidewhich highly inhibits elastase and subtilisin activity, wherein saiddimeric peptide is formed by an intermolecular disulfide bond betweentwo peptides wherein each of the two peptides consist essentially of SEQID NO: 2.